Pili Expression inGeobacter sulfurreducensLacking the Putative Gene for the PilB Pilus Assembly Motor

AbstractMultiple lines of evidence suggest that electrically conductive pili (e-pili) are an important conduit for long-range electron transport inGeobacter sulfurreducens, a common model microbe for the study of extracellular electron transport mechanisms. One strategy to study the function of e-pili has been to delete the gene for PilB, the pilus assembly motor protein, in order to prevent e-pili expression. However, we found that e-pili are still expressed after the gene for PilB is deleted. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-typeG. sulfurreducensor when e-pili are heterologously expressed from theG. sulfurreducenspilin gene inE. coli. Immunogold labeling demonstrated that aG. sulfurreducensstrain expressing e-pili with a His-tag continued to express His-tag labelled e-pili when the PilB gene was deleted. Strains with the PilB gene deleted produced maximum current densities comparable to wild-type controls. These results demonstrate that deleting the gene for PilB is not an appropriate strategy for constructing strains ofG. sulfurreducenswithout e-pili, necessitating a reinterpretation of the results of previous studies that have employed this approach.ImportanceGeobacter sulfurreducensis a model microbe for the study of biogeochemically and technologically significant processes such as the reduction of Fe(III) oxides in soils and sediments; bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity; direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments; and metal corrosion. The phenotypes associated with gene deletions are an important strategy for determining the mechanisms for extracellular electron transfer inG. sulfurreducens. The results reported here demonstrate that a gene deletion previously thought to prevent the expression of electrically conductive pili inG. sulfurreducensdoes not have the intended result. Conductive pili continue to be expressed. This finding is important for interpreting the results of several previous studies on conductive pili function.